Distinct Redox Behaviors of Chloroplast Thiol Enzymes and
their Relationships with Photosynthetic Electron Transport in
Arabidopsis thaliana
1
Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama, 226-8503 Japan
Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo, 102-0075 Japan
3
Imaging Research Division, Bio-AFM Frontier Research Center, Kanazawa University, Kakuma, Kanazawa, 920-1192 Japan
*Corresponding author: E-mail, [email protected]; Fax, +81-45-924-5268.
(Received March 28, 2014; Accepted May 1, 2014)
2
The thiol/disulfide redox network mediated by the thioredoxin (Trx) system in chloroplasts ensures light-responsive
control of diverse crucial functions. Despite the suggested
importance of this system, the working dynamics against
changing light environments remains largely unknown.
Thus, we directly assessed the in vivo redox behavior of
chloroplast Trx-targeted thiol enzymes in Arabidopsis thaliana. In a time-course analysis throughout a day period that
was artificially mimicked to natural light conditions, thiol
enzymes showed a light-dependent shift in redox state, but
the patterns were distinct among thiol enzymes. Notably,
the ATP synthase CF1-g subunit was rapidly reduced even
under low-light conditions, whereas the stromal thiol
enzymes fructose 1,6-bisphosphatase, sedoheptulose 1,7bisphosphatase, and NADP-malate dehydrogenase were
gradually reduced/re-oxidized along with the increase/decrease in light intensity. Photo-reduction of thiol enzymes
was suppressed by the impairment of photosynthetic linear
electron transport using DCMU and 2,5-dibromo-3-methyl6-isopropyl-p-benzoquinone, but sensitivity to the impairment was uneven between CF1-g and other stromal thiol
enzymes. These different dependencies of photo-reduction
on electron transport, rather than the redox state of Trx and
the circadian clock, could readily explain the distinct diurnal
redox behaviors of thiol enzymes. In addition, our results
indicate that the cyclic electron transport around PSI is
also involved in redox regulation of some thiol enzymes.
Based on these findings, we propose an in vivo working
model of the redox regulation system in chloroplasts.
Keywords: Arabidopsis thaliana Chloroplast Photosynthetic electron transport Redox regulation Thiol enzymes
Thioredoxin (Trx).
Abbreviations: AL, actinic light; AMS, 4-acetoamido-40 -maleimidylstilbene-2,20 -disulfonate; AntA, antimycin A; CET,
cyclic electron transport around PSI; CF1, catalytic moiety
of ATP synthase; DBMIB, 2,5-dibromo-3-methyl-6-isopropylp-benzoquinone; FBPase, fructose 1,6-bisphosphatase; Fd, ferredoxin; FTR, ferredoxin-thioredoxin reductase; H2O2, hydrogen peroxide; LET, linear electron transport; MDH, NADPmalate dehydrogenase; NDH, NADPH dehydrogenase; NTA,
nitrilotriacetic acid; PGR5, proton gradient regulation 5; ROS,
reactive oxygen species; SBPase, sedoheptulose 1,7-bisphosphatase; SP, saturating pulse; Trx, thioredoxin.
Regular Paper
Keisuke Yoshida1,2, Yuta Matsuoka1, Satoshi Hara1, Hiroki Konno1,3 and Toru Hisabori1,2,*
Introduction
Proteins located in chloroplasts need to be flexibly and suitably
regulated under fluctuating light environments to ensure efficient chloroplast functions. The electron transport chain in the
thylakoid membrane converts light to ATP and NADPH during
photosynthesis, which are mainly used for the Calvin cycle
reaction in the stroma. Photosynthetic electron transport also
initiates the thiol/disulfide redox cascade by branching electrons at the site of ferredoxin (Fd). Reducing equivalents are
then transferred to specific proteins containing redox-active
Cys residues (referred to as thiol enzymes), allowing modulation
of their enzymatic activities. Therefore, the thiol/disulfide redox
cascade confers several target systems in chloroplasts on the
regulatory way coordinated by light. The key machinery mediating the reducing equivalent transfer from the electron transport chain to thiol enzymes is the thioredoxin (Trx) system
composed of Fd-Trx reductase (FTR) and Trx.
Trx is a small ubiquitous protein with an active site containing one pair of Cys residues that undergoes reversible reduction
and oxidation. Trx was first identified in Escherichia coli in 1964
as a ribonucleotide reductase cofactor (Laurent et al. 1964). In
the 1970s, members involved in the Trx system, such as Trx and
FTR, were identified in chloroplasts (Buchanan and Wolosiuk
1976, Schürmann et al. 1976, Wolosiuk and Buchanan 1977).
It was further demonstrated that four Calvin cycle
Plant Cell Physiol. 55(8): 1415–1425 (2014) doi:10.1093/pcp/pcu066, available online at www.pcp.oxfordjournals.org
! The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Plant Cell Physiol. 55(8): 1415–1425 (2014) doi:10.1093/pcp/pcu066 ! The Author 2014.
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K. Yoshida et al.
enzymes, namely fructose 1,6-bisphosphatase (FBPase), sedoheptulose 1,7-bisphosphatase (SBPase), NADP-glyceraldehyde 3phosphate dehydrogenase, and phosphoribulokinase are activated when the Trx system is reconstituted in vitro (for a recent
review, see Michelet et al. 2013). These reports led to the
establishment of the current basic concept regarding the
chloroplast Trx system; Trx receives reducing equivalents
from the light-driven photosynthetic electron transport chain
through FTR and subsequently reduces specific disulfide
bridges on thiol enzymes. Further study using intact chloroplasts indicated that this machinery is physiologically functional
(Crawford et al. 1989).
Proteins subjected to Trx-dependent thiol modulation are
not restricted to Calvin cycle enzymes. Early studies showed
that Trx participates in the activation of ATP synthase
(McKinney et al. 1979). Thereafter, two adjacent Cys residues
localized in the central axis of the CF1-g subunit were revealed
to have a critical role in redox regulation of ATP synthase (Nalin
and McCarty 1984, Miki et al. 1988). Proteins mediating other
metabolic pathways such as NADP-malate dehydrogenase
(MDH) involved in the malate valve have been also known to
be redox-regulated through the Trx system (Scheibe 1981) but
understanding of the Trx-targeted thiol enzyme remained limited until 2000. Information on the Trx target protein was drastically broadened in 2001 by the development of methodology
for capturing Trx-interacting proteins (Motohashi et al. 2001,
Yano et al. 2001). Accompanied by advanced proteomic techniques, these strategies have been used to explore Trx target
candidates not only in chloroplasts but also in other cellular
compartments including cytosol (Yamazaki et al. 2004) and
mitochondria (Balmer et al. 2004, Yoshida et al. 2013). This
has lead to the discovery of Trx-linked proteins associated
with a broad spectrum of cellular processes (see Montrichard
et al. 2009 and Lindahl et al. 2011 for reviews).
The plant Trx system is characterized by divergent Trx subtypes. At least 20 Trx genes are divided into seven classes (f-, m-,
h-, o-, x-, y-, and z-type) in Arabidopsis thaliana. Among them,
as many as five classes (f-, m-, x-, y-, and z-type) are targeted to
chloroplasts. Trx-f and Trx-m were initially identified as the
light-dependent activators that preferentially reduce FBPase
and MDH, respectively (Schürmann et al. 1981). Trx-x and -y
were newly discovered through the increased availability of
plant genome information and efficiently donate reducing
equivalents to the anti-oxidant defense system (Collin et al.
2003, Collin et al. 2004). Trx-z was recently identified as a
novel Trx required for chloroplast development by regulating
transcription in chloroplast genes (Arsova et al. 2010). Besides
these Trx subtypes, NADPH-Trx reductase C is also localized to
chloroplasts (Serrato et al. 2004) and mediates redox regulation
of several processes, including anti-oxidant defense (Pérez-Ruiz
et al. 2006), starch synthesis (Michalska et al. 2009), and tetrapyrrole metabolism (Richter et al. 2013). These findings suggest
that chloroplasts host a complex redox network, although the
functional diversity is yet to be fully clarified (see König et al.
2012 and Serrato et al. 2013 for reviews).
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As summarized above, many previous efforts with a long
history have substantially contributed to the knowledge of
the Trx-mediated redox pathway and its possible target proteins in chloroplasts. Thanks to the direct determination of
enzyme structure, molecular mechanisms of the redox regulation system are becoming increasingly apparent (Michelet et al.
2013). However, because the current understanding has been
achieved by biochemical in vitro assays or proteomics-based
analyses, it is nearly completely unclear how the chloroplast
Trx system works in vivo. Using a method to visualize the
in vivo redox state, we recently demonstrated that the ATP
synthase CF1-g subunit in spinach leaves shows a drastic shift
in redox state throughout the day (Konno et al. 2012). Here, we
performed a comparative study of the in vivo redox behaviors of
several thiol enzymes in A. thaliana to obtain more comprehensive insight into the chloroplast redox regulation system in
living cells. We also addressed these regulatory mechanisms
with a focus on the association with photosynthetic electron
transport. We provide evidence that redox behaviors to changing light environments are not uniform among thiol enzymes,
which results from different electron transport efficiency
relationships.
Results
Chloroplast thiol enzymes show distinct diurnal
redox behaviors
We first assessed the diurnal dynamics of in vivo thiol modulation. For this purpose, a growth chamber was programmed
to mimic light conditions in the field (Fig. 1A). Light intensity was gradually and almost linearly elevated under programmed conditions and reached approximately 350 mmol
photons m-2 s-1 at 5 h after the onset of illumination. Light intensity was then gradually attenuated and completely turned
off at 11 h after the onset of illumination. During this period,
Arabidopsis wild-type plants were placed in the chamber
and sequentially sampled, followed by determining the reduction level of each thiol enzyme and Trx (see Materials and
Methods).
Our method for visualizing the in vivo redox state is based on
the difference in protein mobility on non-reducing SDS-PAGE
between the reduced [regulatory Cys modified with 4-acetoamido-40 -maleimidylstilbene-2,20 -disulfonate (AMS)] and oxidized (unmodified) forms (Motohashi et al. 2001). As shown
in Fig. 1B, we observed a clear band shift in several thiol enzymes depending on the change in light environment.
Interestingly, the observed redox behaviors were not uniform
among thiol enzymes (Fig. 1C). The ATP synthase CF1-g subunit showed a rapid shift from the oxidized to the reduced form
at the very beginning after turning on the light when light
intensity was still weak. CF1-g was maintained in almost a
fully reduced state until the light was completely turned off.
In contrast, FBPase and SBPase were reduced slowly concomitant with the increase in light intensity, followed by gradual
Plant Cell Physiol. 55(8): 1415–1425 (2014) doi:10.1093/pcp/pcu066 ! The Author 2014.
Redox dynamics of chloroplast thiol enzymes
300
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B
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Light intensity
(μmol photons m-2 s-1)
A
CF1-γ
Red
Ox
FBPase
Red
Ox
SBPase
Red
Ox
Trx-m2
Red
Ox
Trx-f 2
Red
Ox
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C
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SBPase
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MDH
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Time after dark period (h)
0.5
Trx-m2
Trx-f2
0.8
0
Reduction level
0.4
Red
Ox
0.2
0
RbcL
0
2
4
6
8
10
12
Time after dark period (h)
Fig. 1 Diurnal redox dynamics of several thiol enzymes and thioredoxins (Trxs). (A) Change in the light intensity under programmed conditions.
(B) Visualization of diurnal change in the redox state of the ATP synthase CF1-g subunit, fructose 1,6-bisphosphatase (FBPase), sedoheptulose 1,7bisphosphatase (SBPase), Trx-m2, and Trx-f2. Proteins were extracted from the leaves at the times indicated and the redox state of each enzyme
was visualized as described in Materials and Methods. Each upper band corresponds to the regulatory Cys-modified reduced form (Red), while
the lower band corresponds to the regulatory Cys-unmodified oxidized form (Ox). The same amount of leaf total protein was loaded into each
lane and the Rubisco large subunit (RbcL) was stained with Ponceau S as a loading control. (C) Diurnal change in the reduction level of the ATP
synthase CF1-g subunit, FBPase, SBPase, Trx-m2, and Trx-f2. The reduction level was quantified as the ratio of the reduced form to the total.
Immunoblotting analysis was repeated six times using three different sample preparations (two analyses per sample preparation) and the
mean ± SD is shown. (D) Visualization of diurnal change in the redox state of NADP-malate dehydrogenase (MDH). Experiments were performed
as described in (B).
re-oxidation under decreasing light intensity. Although the induction pattern of photo-reduction was similar between
FBPase and SBPase, the saturating reduction level was different;
FBPase reached >80% of full reduction, whereas SBPase reached
only approximately 60%. MDH did not show a clear band shift
between oxidized and reduced forms (Fig. 1D). This was possibly due to the fact that more than one pair of Cys residues
are involved in the redox regulation of MDH (Miginiac-Maslow
et al. 2000). Nevertheless, it was apparent that MDH was
converted gradually from an oxidized to a reduced form by
increasing light intensity, and vice versa by decreasing light
intensity.
We also examined the redox state of two Trx isoforms, Trx-m2
and Trx-f2 (Fig. 1B, C). Three discriminative bands were observed
for Trx-f2, which may have been related to the cross-reaction with
Trx-f1 or the glutathionylation of the additional Cys conserved in
Trx-f (Michelet et al. 2005). As the most upper band emerged
after illumination, the reduction level of Trx-f2 was quantified on
the assumption that the upper band corresponded to the
reduced form. Partial conversion to the reduced form was
observed for both in Trx-m2 and Trx-f2 upon illumination.
However, these responses were not so drastic toward
the change in light intensity and the reduction levels were kept
almost stable (<50% of full reduction) throughout a day period.
The diurnal redox behaviors of chloroplast thiol
enzymes are linked to light intensity, not the
circadian clock
We next examined whether circadian clock-dependent regulation is involved in diurnal dynamics of thiol modulation.
Following a gradual increase, light intensity was maintained at
a saturating level until 11 h after the onset of illumination
(Fig. 2A). Under such prolonged light conditions, the reduction
levels of all thiol enzymes and Trxs examined here were kept at
saturating states (Fig. 2B, C), indicating that diurnal redox behaviors of thiol enzymes are absolutely linked to light intensity,
not the circadian clock.
Plant Cell Physiol. 55(8): 1415–1425 (2014) doi:10.1093/pcp/pcu066 ! The Author 2014.
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K. Yoshida et al.
400
Light intensity
(μmol photons m-2 s-1)
A
Photosynthetic linear electron transport efficiency
differentially affects the photo-reduction pattern
of each thiol enzyme
300
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Trx-f2
Red
Ox
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Ox
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Time after dark period (h)
CF1-γ
Red
Ox
MDH
RbcL
RbcL
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Reduction level
C
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FBPase
SBPase
0
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Trx-f2
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The Trx system is associated with light through the photosynthetic electron transport chain in the thylakoid membrane.
Photosynthetic electron transport consists of linear electron
transport (LET) from water to NADP+ and cyclic electron transport around PSI (CET). Although the contribution of CET in
photosynthesis is still a matter of intense debate, several lines of
evidence show that this pathway is necessary for driving thermal dissipation of excess light energy and avoiding over-reduction of PSI (for a review, see Shikanai 2007). In order to gain
insights into the relationship between redox dynamics and the
efficiency of each photosynthetic electron transport, we examined the photo-reduction patterns of thiol enzymes under conditions where LET and CET are individually impaired.
Leaves were treated with DCMU (inhibitor of electron transfer between QA and QB in PSII) or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB; inhibitor of electron transfer
between plastoquinone and the cytochrome b6/f complex) to
restrict LET. The inhibitory effects were then validated by monitoring Chl fluorescence (Fig. 3A). When leaves were treated
with 10 mM DCMU or DBMIB, the basal level of Chl fluorescence under actinic light (AL) was significantly elevated.
However, a saturating pulse (SP) caused a transient increase
in Chl fluorescence, indicating that LET was still partially functional. In contrast, when each inhibitor was used at 100 mM, the
basal level of Chl fluorescence reached nearly a maximum level
and SP no longer excited Chl fluorescence. It was thus confirmed that LET was completely impaired in the presence of
100 mM of each inhibitor.
Inhibitor-treated leaves were illuminated at two different
intensities (low light: 30 mmol photons m-2 s-1; high light:
800 mmol photons m-2 s-1), followed by visualization of the
redox state of the thiol enzymes (Fig. 3B). CF1-g in the control
treatment was fully reduced under both light regimes, which
coincided with the data shown in Fig. 1. In the presence of
10 mM inhibitor in which LET was partly restricted, about half of
the CF1-g was reduced upon illumination. Interestingly, while
CF1-g was partially photo-reduced in the presence of 100 mM
DCMU, it was completely kept in the oxidized form in the
presence of 100 mM DBMIB. Given that DBMIB inhibits both
LET and CET, this result suggests that CET is also involved in the
redox regulation of CF1-g (see below). In contrast to CF1-g,
photo-reduction of other thiol enzymes, FBPase, SBPase, and
MDH, was completely suppressed by DCMU or DBMIB even at
Time after dark period (h)
Fig. 2 Redox behaviors of several thiol enzymes and thioredoxins
(Trxs) under prolonged light conditions. (A) Change in the light intensity under programmed conditions. (B) Visualization of the change
in the redox state of the ATP synthase CF1-g subunit, FBPase, SBPase,
MDH, Trx-m2, and Trx-f2 during prolonged light conditions.
Experiments were performed as described in the legend of Fig. 1.
(C) Change in the reduction level of the ATP synthase CF1-g subunit,
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Fig. 2 Continued
FBPase, SBPase, Trx-m2, and Trx-f2 during prolonged light conditions. The
reduction level was quantified as the ratio of the reduced form to the
total. Immunoblotting analysis was repeated six times using three different
sample preparations (two analyses per sample preparation) and the
mean ± SD is shown.
Plant Cell Physiol. 55(8): 1415–1425 (2014) doi:10.1093/pcp/pcu066 ! The Author 2014.
Redox dynamics of chloroplast thiol enzymes
A
100 μM DCMU
10 μM DCMU
Control
10 mM (Fig. 3B). Similarly, photo-reduction of Trx-m2 and
Trx-f2 was sensitively inhibited by partial impairment of LET
(Fig. 3B).
1 min
1
0.8
0.6
Cyclic electron transport around PSI is involved in
the redox regulation of some thiol enzymes
Chl fluorescence
0.4
0.2
0
AL
SP
100 μM DBMIB
10 μM DBMIB
1
0.8
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0
10 μM DBMIB
100 μM DBMIB
10 μM DCMU
100 μM DCMU
Control
10 μM DBMIB
800
100 μM DBMIB
10 μM DCMU
Control
Dark
100 μM DCMU
30
B Light intensity:
CF1-γ
Red
Ox
FBPase
Red
Ox
SBPase
Red
Ox
MDH
Red
Ox
Trx-m2
Red
Ox
Trx-f2
Red
Ox
RbcL
Fig. 3 Effects of linear electron transport (LET) impairment on photoreduction of several thiol enzymes and thioredoxins (Trxs). (A) Traces
of Chl fluorescence in control and LET-impaired leaves. Chl fluorescence is represented as a relative value to the maximum level (Fm).
DCMU and DBMIB were used at 10 mM or 100 mM to inhibit LET. At
the time indicated by the red arrow, leaves were illuminated with
actinic light (AL: 22 mmol photons m-2 s-1). At the time indicated by
the blue arrow, leaves were illuminated with a saturating pulse (SP).
(B) Photo-reduction patterns of the ATP synthase CF1-g subunit,
FBPase, SBPase, MDH, Trx-m2, and Trx-f2 in control and
We next addressed whether CET has an impact on thiol modulation. For this purpose, a CET inhibitor antimycin A (AntA)
was applied to leaves, and the effects on photo-reduction of
thiol enzymes were examined (Fig. 4). The reduction levels of
CF1-g and FBPase were slightly higher in AntA-treated leaves
than those in the control under low-light conditions.
Conversely, the reduction level of FBPase was lowered when
leaves were treated with AntA under high-light conditions.
AntA did not affect photo-reduction of SBPase under either
light condition.
AntA is also a typical inhibitor of electron transfer in the
mitochondrial respiratory chain, and mitochondrial metabolism supports photosynthesis through organelle crosstalk in
illuminated leaves (Noguchi and Yoshida 2008). Thus, we
could not rule out the possibility that the results shown in
Fig. 4 were caused by impairment of the respiratory chain
rather than CET. Therefore, the involvement of CET in thiol
modulation was also validated using the proton gradient regulation5 (pgr5) mutant, which fails to perform AntA-sensitive CET
(Munekage et al. 2002). The photo-reduction patterns of the
thiol enzymes were compared between wild-type and pgr5 at
several light intensities (Fig. 5A, B). Under low-light conditions,
the reduction levels of CF1-g and FBPase were slightly higher in
pgr5 than in the wild type. FBPase was almost fully reduced in
the wild-type under high-light conditions, whereas 20–30%
FBPase was present in the oxidized form in pgr5. Genetic impairment of CET did not significantly affect photo-reduction of
SBPase as was the case with AntA treatment.
CET is also mediated by another pathway catalyzed by the
AntA-insensitive NADPH dehydrogenase (NDH) complex. This
pathway is not functional in the chlororespiratory reduction2
(crr2) mutant due to the lack of proper NDH complex formation
(Hashimoto et al. 2003). By treating mutants with AntA, we
assessed the difference in the contribution of PGR5- and NDHdependent CET to thiol modulation under high-light conditions
(1,100 mmol photons m-2 s-1, Fig. 5C). Photo-reduction of CF1-g
was not affected by any impairments in CET. FBPase was partly
present in the oxidized form in pgr5, which was no longer affected by AntA due to the intrinsic lack of an AntA-sensitive
pathway. FBPase was fully photo-reduced in crr2 in the absence
of AntA, but was partly oxidized by AntA treatment. This
Fig. 3 Continued
LET-impaired leaves. After treatment, the redox state of each enzyme was
visualized as described in the legend of Fig. 1. Light intensity during the
treatment is indicated above the image (30 and 800 mmol photons m-2 s-1).
Experiments were repeated three times using different sample preparations and the representative results are shown.
Plant Cell Physiol. 55(8): 1415–1425 (2014) doi:10.1093/pcp/pcu066 ! The Author 2014.
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K. Yoshida et al.
FBPase
SBPase
MDH
100 μM AntA
Control
100 μM AntA
800
CF1-γ
B
Red
Ox
Red
Ox
Red
Ox
Red
Ox
1
Reduction level
CF1-γ
10 μM AntA
Control
30
10 μM AntA
Light
:
intensity
Dark
A
FBPase
c
a
c
SBPase
e
c
d
b
ab
d
0.8
b
0.6
0.4
a
b
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a
a
a
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100
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b
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100
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Light intensity:
0
10
100
30
0
10
800
100
0
10
30
100
0
10
800
100
30
0
800
RbcL
Fig. 4 Effects of cyclic electron transport around PSI (CET) impairment by antimycin A (AntA) treatment on photo-reduction of several
thiol enzymes. (A) Photo-reduction patterns of the ATP synthase CF1-g subunit, FBPase, SBPase, and MDH in control and CET-impaired leaves.
AntA was used at 10 mM or 100 mM to inhibit CET. Experiments were performed as described in the legend of Fig. 3. (B) Effects of AntA
treatment on the reduction level of the ATP synthase CF1-g subunit, FBPase, and SBPase. The reduction level was quantified as the ratio of the
reduced form to the total. Immunoblotting analysis was repeated six times using three different sample preparations (two analyses per sample
preparation) and the mean ± SD is shown. Different letters denote significant differences among treatments (P < 0.05, Tukey-Kramer multiple
comparison test).
response was similar to that of the wild-type. Taken together,
NDH-dependent CET did not significantly affect thiol modulation, whereas PGR5-dependent CET had some impact. These
results are in accordance with the findings that PGR5-deficient
plants, but not NDH-deficient plants, exhibit altered overall electron transport properties (e.g. Munekage et al. 2004).
Discussion
Chloroplast redox regulation through the Trx system has been
generally acknowledged as an important system for controlling
diverse functions in response to light. However, while our
understanding of redox regulation has grown widely at the
biochemical and structural levels, a quite fundamental but
physiologically important question of how this system responds
to light in vivo has remained unanswered. Thus, we assessed the
redox dynamics of several thiol enzymes in illuminated leaves
and their regulatory mechanisms.
Distinct diurnal redox behaviors between ATP
synthase CF1-c subunit and stromal thiol enzymes
are primarily attained by different dependencies
on LET
As shown in Fig. 1, we observed distinct diurnal redox behaviors of thiol enzymes. The most remarkable feature was found
in the ATP synthase CF1-g subunit, which was almost fully
reduced even under low-light conditions. Using the electrochromic shift assay, Kramer et al. (1990) reported that ATP
1420
synthase is fully activated even under quite low-light conditions where the CO2 fixation rate is still not saturated. As ATP
synthase is regulated by a multi-step mechanism (for a review,
see Hisabori et al. 2013), it was unclear only from their study
whether this activation was linked to thiol modulation of CF1g. Our present data directly visualizing the in vivo redox state
strongly indicate that rapid photo-reduction of CF1-g enables
ATP synthase to be activated even under light-limited conditions. In contrast, the stromal enzymes FBPase, SBPase, and
MDH were gradually photo-reduced/re-oxidized concomitant
with the increase/decrease in the light intensity. What factor
controls these distinct responses? In general, LET in the thylakoid membrane is accelerated in parallel with the increase in
light intensity and reaches the saturating rate under high-light
conditions (e.g. Yoshida et al. 2011), although we could not
monitor the diurnal pattern of LET efficiency due to the
technical limit of the measurement. In experiments using
LET inhibitors, we found that CF1-g was photo-reduced
even when LET was partially restricted, whereas photoreduction of other stromal thiol enzymes was completely
abolished under the same conditions (Fig. 3). These results
indicate that photo-reduction of CF1-g can be sensitively triggered in response to subtle induction of LET, while others
cannot be photo-reduced until LET is substantially activated
(Fig. 6).
The transcript levels of Trx genes fluctuate through circadian regulation (Barajas-López et al. 2011) or in response to the
accumulation of photosynthetic products (Barajas-López et al.
2012). Furthermore, recent reverse-genetic studies have
Plant Cell Physiol. 55(8): 1415–1425 (2014) doi:10.1093/pcp/pcu066 ! The Author 2014.
Redox dynamics of chloroplast thiol enzymes
Fig. 5 Difference of the redox behaviors of several thiol enzymes between wild-type (WT) plants and cyclic electron transport around PSI (CET)
mutants. (A) Photo-reduction patterns of the ATP synthase CF1-g subunit, FBPase, SBPase, and MDH in WT plants and the pgr5 mutants. Each
plant was placed under the light conditions indicated above the image (0-650 mmol photons m-2 s-1, 25 C) for 30 min. Then, the redox state of
each enzyme was visualized as described in the legend of Fig. 1. (B) Comparison of the reduction level of the ATP synthase CF1-g subunit, FBPase,
and SBPase between WT plants and pgr5 mutants. The reduction level was quantified as the ratio of the reduced form to the total.
Immunoblotting analysis was repeated six times using three different sample preparations (two analyses per sample preparation) and the
mean ± SD is shown. Asterisks denote a significant difference between WT and pgr5 (*P < 0.05, **P < 0.01, Student’s t-test). (C) Effects of
antimycin A (AntA) treatment on the reduction level of the ATP synthase CF1-g subunit and FBPase in WT, pgr5, and crr2 mutants. After the
treatment, the redox state of each enzyme was visualized as described in the legend of Fig. 1. Light intensity during the treatment was set at 0
(dark) or 1,100 mmol photons m-2 s-1.
demonstrated that manipulating Trx accumulation level leads
to a change in the redox state of some thiol enzymes. For example, disruption of Trx-f1 gene results in impaired photoreduction of ADP-glucose pyrophosphorylase in Arabidopsis
(Thormählen et al. 2013). Therefore, the amount or redox
states of Trxs are also candidates for determining diurnal
redox behavior of thiol enzymes. However, a clear change in
protein abundance of Trx-m2 and Trx-f2 was not observed
throughout a day period (Fig. 1). More notably, the redox
states of Trxs did not shift drastically regardless of light intensity
and were maintained at <50% of full reduction (Fig. 1). Given
that the impairment of LET abolished the photo-reduction of
Trxs (Fig. 3), Trx must be located on a route necessary for
transferring reducing equivalents from photosynthetic electron
transport to thiol enzymes. However, the results shown in Fig. 1
suggest that the amount and redox state of Trx are not rate
limiting for regulating diurnal redox behaviors of thiol enzymes,
at least under non-stressful conditions. We also addressed
whether circadian clock-dependent regulation is directly
involved in thiol modulation (Fig. 2). The results clearly demonstrated that diurnal redox behaviors of thiol enzymes are not
regulated by the circadian clock, but are strictly linked to light
intensity. Taken together, these results allowed us to conclude
that different dependencies on LET primarily confer CF1-g
and stromal thiol enzymes to distinct diurnal redox behaviors
(Fig. 6).
An intriguing question is then raised about the mechanism
of different dependencies on LET between CF1-g and stromal
thiol enzymes. It appears difficult to explain this mechanism
only by a difference in midpoint redox potential (Em), because
the reported Em values of thiol enzymes including CF1-g are not
much different from that of Trx (Kramer and Crofts 1989,
Hirasawa et al. 1999, Hutchison et al. 2000). The most plausible
hypothesis is that it results from different localization in chloroplasts. When LET is activated, reducing equivalents may be
easily transferred to CF1-g (peripherally residing in the thylakoid
membrane) more than other thiol enzymes (drifting in the
stroma). In support of this idea, the membrane-bound
Plant Cell Physiol. 55(8): 1415–1425 (2014) doi:10.1093/pcp/pcu066 ! The Author 2014.
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K. Yoshida et al.
Redox state of thiol enzymes
Ox
SBPase
Red
SBPase
FBPase
MDH
FBPase
Competing for electrons
with the Trx system
Trx
Trx
ROS
Fd
Avoiding PSI
over-reduction
Trx
Fd
CF1-γ
PSI
MDH
FBPase
MDH
Fd
PSII
SBPase
CF1-γ
PSII
Dark
PSII
PSI
ATP synthase
CF1-γ
PSI
ATP synthase
Low light
ATP synthase
High light
Fig. 6 Simplified in vivo working model of the chloroplast redox regulation system. Black, red, and blue arrows indicate photosynthetic electron
transport in the thylakoid membrane, the transfer of reducing equivalents by the Trx system, and ROS-derived oxidation, respectively. The
magnitude of each pathway is roughly represented as follows: dotted arrows: low, solid thin arrows: middle, solid bold arrows: high. Possible role
of cyclic electron transport around PSI (CET) is also described in the box.
machinery for transferring reducing equivalents has been suggested (Anderson and Avron 1976). Furthermore, proteomics
analyses have demonstrated that some Trxs are located adjacent to the thylakoid membrane (Peltier et al. 2002, Friso et al.
2004). Such components may enable CF1-g to be rapidly photoreduced in response to LET, but this possibility needs to be
experimentally validated. It is noteworthy that the proximity
seems to be a key factor for thiol/disulfide exchange reaction in
mammalian cells (Gutscher et al. 2009).
Distinct redox behaviors were found even between
FBPase and SBPase, both of which are stromal enzymes; the
saturating photo-reduction level in FBPase was higher than
that in SBPase (Fig. 1). The mechanisms underlying this
difference could not be elucidated by present study.
However, SBPase, but not FBPase, was recently determined
to be a hydrogen peroxide (H2O2)-sensitive protein based
on proteomics-based screening (Muthuramalingam et al.
2013). As reactive oxygen species (ROS), including H2O2,
inevitably accumulate along with the increase in light intensity, ROS-derived oxidation must be a potent factor for lowering the apparent saturating photo-reduction level of SBPase
(Fig. 6). Nevertheless, the oxidation mechanisms of thiol enzymes need to be addressed with paying attention to other
oxidant candidates such as the oxidized forms of Trx and
glutathione (Wolosiuk and Buchanan 1977, Scheibe and
Anderson 1981).
FBPase under low-light conditions (Figs. 4, 5). These results
indicate that the Trx system competes with CET for electrons
transferred to Fd under light-limited conditions (Fig. 6). This
possibility is supported by our previous study showing that the
MDH activation state is higher in the pgr5 mutant than that in
wild-type plants under low-light conditions (Yoshida et al.
2007). In contrast, impairment of PGR5-dependent CET lowered the reduction level of FBPase under high-light conditions
(Figs. 4, 5). As mentioned above, ROS-mediated oxidation of
SBPase may occur even in the wild-type under high-light conditions. It is possible that ROS generation is further elevated by
blocking CET resulting in PSI over-reduction, which promotes
re-oxidation of FBPase, in addition to SBPase (Fig. 6). In support
of this idea, it was recently shown by estimating the protein
carbonylation level that oxidation power is liable to be exerted
in the pgr5 mutant more than in the wild-type (Suorsa et al.
2012). Therefore, CET may be indirectly involved in thiol modulation by suppressing over-reduction of PSI and thereby controlling ROS generation under high-light conditions. These
scenarios should be dissected in more detail, but there is little
doubt that CET has a significant role in fine tuning of the redox
regulation system, and that its mode varies depending on light
intensity. It should be noted that the efficiency of CET itself may
be under redox regulation through the Trx system (Hertle et al.
2013).
Future perspectives
How is CET involved in redox regulation of
chloroplast thiol enzymes?
Another intriguing finding in this study is that CET was
involved in redox regulation of some thiol enzymes (Figs. 4,
5). Courteille et al. (2013) demonstrated that Trx-m4 exerts
negative control on CET. Our results showed that both chemical and genetic impairments of PGR5-dependent CET slightly
but significantly elevated the reduction level of CF1-g and
1422
In this study, we visualized the in vivo diurnal redox dynamics of
several thiol enzymes in chloroplasts. Notably, redox behaviors
were distinct among thiol enzymes, which was closely associated with electron transport efficiency. These findings provide
insight into how chloroplasts adjust their own functions to
changing light environments. In order to understand chloroplast redox dynamics more deeply, it is important to clarify
other redox-related parameters such as fluctuation of
Plant Cell Physiol. 55(8): 1415–1425 (2014) doi:10.1093/pcp/pcu066 ! The Author 2014.
Redox dynamics of chloroplast thiol enzymes
NADPH/NADP+ ratio and the engagement of anti-oxidant
defense systems. Besides our present study, the in vivo redox
regulation system, including the distribution of Trx with
respect to thiol enzymes (Anderson et al. 2008) and the specific role of each Trx subtype (Chi et al. 2008, Laugier et al.
2013, Thormählen et al. 2013) has been recently addressed,
but is only starting to emerge. Further investigation of
these subjects will promote our understanding of the redox
network ensuring light-responsive thiol modulation in
chloroplasts.
Materials and Methods
Plant material
A. thaliana wild-type plants (Col-gl1), the pgr5 mutant, and the
crr2 mutant (provided courtesy of Prof. T. Shikanai) were grown
in soil in a controlled growth chamber (80–90 mmol photons
m-2 s-1, 22 C, 16 h day/8 h night) for 4 weeks. For the analysis
shown in Figs 1 and 2, plants were transferred to a programmed chamber at the end of night period (i.e. just before
the onset of program).
Preparation of antibodies
The spinach CF1-g antibody used in our previous study showed
low affinity to the regulatory Cys-modified reduced form compared with the unmodified oxidized form, possibly because this
antibody was raised against the partial polypeptide including
the regulatory Cys-containing region (Konno et al. 2012). Thus,
a new antibody was generated to improve the accuracy of
estimating the CF1-g redox state. Recombinant Arabidopsis
CF1-g (His-tagged at the C terminus) was prepared as follows.
The ATPC1 (At4g04640) gene fragment encoding the mature
protein region (Ala51-Val373) was cloned into the pET23a expression vector (Novagen, Gibbstown, NJ, USA). The plasmid
was transformed to E. coli BL21 (DE3), and the CF1-g protein
was expressed with 0.5 mM IPTG. E. coli cells were disrupted
with a French pressure cell (5501-M, Ohtake Works, Tokyo,
Japan). CF1-g, mainly expressed in the inclusion body, was solubilized with 8 M urea in 50 mM phosphate buffer (pH 8.0). After
centrifugation at 20,000 g for 5 min, the resulting supernatant was loaded onto a Ni-nitrilotriacetic acid (NTA) affinity
column (Qiagen, Valencia, CA, USA). CF1-g was eluted by lowering the pH to 5.8, which was used as the antigen to prepare
the specific antibody.
The MDH, Trx-f2, and Trx-m2 recombinant proteins were
also prepared for use as antigens. MDH and Trx-f2 were expressed as His-tagged proteins at the C-terminus. The
Arabidopsis MDH (At5g58330) gene fragment encoding the mature protein region (Val55-Val443) was cloned into the pET23d
expression vector (Novagen) to prepare MDH. The Arabidopsis
Trx-f2 (At5g16400) and Trx-m2 (At4g03520) gene fragments
encoding the mature protein region (Glu71-Gly185 for Trx-f2
and Glu77-Pro186 for Trx-m2) were individually cloned into
the pET23a expression vector to prepare Trx-f2 and Trx-m2,
respectively. Each plasmid was transformed to E. coli BL21
(DE3), and the proteins were expressed with 0.5 mM IPTG. E.
coli cells were disrupted by sonication. After centrifugation
(125,000 g for 40 min), the resulting supernatant was used
to purify the proteins of interest. Purification was performed
by combining Ni-NTA affinity chromatography, anion exchange chromatography [using DEAE-Toyopearl 650M
column (Tosoh, Tokyo, Japan) and QAE-Toyopearl 550C
column (Tosoh)], and hydrophobic interaction chromatography [using Butyl-Toyopearl 650M (Tosoh)] as described in
Yoshida et al. (2013).
Protein purification was conducted at 4 C. The FBPase antibody was prepared in a previous study (Konno et al. 2012). The
SBPase antibody was provided courtesy by Dr. M. Tamoi (Kinki
University).
Visualization of the in vivo redox state of thiol
enzymes
The in vivo reduction level of chloroplast thiol enzymes was
determined according to Konno et al. (2012) with modifications. Plants were placed directly in liquid nitrogen and ground
using a mortar and pestle. Proteins were extracted in the SDS
sample buffer [2% (w/v) SDS, 62.5 mM Tris-HCl (pH 6.8), 7.5%
(v/v) glycerol and 0.01% (w/v) bromophenol blue] containing
the protease inhibitor cocktail Complete (Roche, Manheim,
Germany) and the specific thiol-labeling reagent AMS
(Invitrogen, Carlsbad, CA, USA). The samples were incubated
for 60 min at room temperature to complete the labeling of
thiol groups with AMS. Non-reducing SDS-PAGE and immunoblotting were performed using a standard method as described
in (Yoshida et al. 2007). Protein concentration was determined
with a BCA protein assay (Pierce, Rockford, IL, USA).
Inhibitor treatments
Inhibitor treatments to impair photosynthetic electron transport were performed according to Yoshida and Noguchi (2009)
with slight modifications. Leaves were excised from plants and
vacuum-infiltrated for 5 min with several inhibitor solutions of
DCMU, DBMIB, and AntA. As a control, leaves were treated
with 1% (v/v) ethanol (used as a solvent for the inhibitors).
After vacuum infiltration, leaves submerged in the inhibitor
solutions were placed under the indicated light conditions for
30 min. The temperature during treatments was set to 25 C.
The inhibitory effects of DCMU and DBMIB on photosynthetic
electron transport were confirmed by Chl fluorescence measurements. Chl fluorescence was monitored using mini-PAM
(Walz, Effeltrich, Germany).
Statistical analysis
Statistical analyses were conducted with Student’s t-test using
Microsoft Excel (Microsoft, Inc., Redmond, WA, USA) and
Tukey-Kramer’s multiple comparison test using SPSS 12.0J software (SPSS, Inc., Chicago, IL, USA).
Plant Cell Physiol. 55(8): 1415–1425 (2014) doi:10.1093/pcp/pcu066 ! The Author 2014.
1423
K. Yoshida et al.
Funding
This work was supported in part by the Core Research of
Evolutional Science and Technology program (CREST) from
the Japan Science and Technology Agency (JST) and a Grantin-Aid for Scientific Research (grant number 24870010 to K.Y.)
from the Japan Society for the Promotion of Science.
Acknowledgments
We thank T. Shikanai and M. Tamoi for the generous donation
of Arabidopsis seeds and SBPase antibody, respectively. We also
thank H. Ohta, S. Masuda and R. Sato for use of mini-PAM.
Disclosures
The authors have no conflicts of interest to declare.
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